Host-dependent resistance of Group A Streptococcus to sulfamethoxazole mediated by a horizontally-acquired reduced folate transporter

Described antimicrobial resistance mechanisms enable bacteria to avoid the direct effects of antibiotics and can be monitored by in vitro susceptibility testing and genetic methods. Here we describe a mechanism of sulfamethoxazole resistance that requires a host metabolite for activity. Using a combination of in vitro evolution and metabolic rescue experiments, we identify an energy-coupling factor (ECF) transporter S component gene (thfT) that enables Group A Streptococcus to acquire extracellular reduced folate compounds. ThfT likely expands the substrate specificity of an endogenous ECF transporter to acquire reduced folate compounds directly from the host, thereby bypassing the inhibition of folate biosynthesis by sulfamethoxazole. As such, ThfT is a functional equivalent of eukaryotic folate uptake pathways that confers very high levels of resistance to sulfamethoxazole, yet remains undetectable when Group A Streptococcus is grown in the absence of reduced folates. Our study highlights the need to understand how antibiotic susceptibility of pathogens might function during infections to identify additional mechanisms of resistance and reduce ineffective antibiotic use and treatment failures, which in turn further contribute to the spread of antimicrobial resistance genes amongst bacterial pathogens.

Standard preparation. Reference standards for each standard was from suppliers as indicated in Supplementary Table 6. Stock solutions were prepared at 1 mg/mL in DMSO, and further diluted to 10 µg/mL in methanol containing 0.1 % butylated hydroxytolunene (BHT) for LC-MS analysis.
Sample preparation. Media samples were filtered using a 3 kDa MWCO filter (Millipore Centriprep) and either directly injected for the detection of targeted tetrahydrofolic acid metabolites, or diluted 1 in 10 (v/v) with LC-MS grade water for untargeted profiling. Preparation was identical for both methods.
LC-MS method. Chromatographic separation was performed on a Waters Acquity I-class UPLC system (Waters, Wilmslow, UK), using a Waters Cortecs T3, 100mm x 2.1mm x 1.6 µm (Waters, Wilmslow, UK) kept at 40 ºC. Mobile phase A consisted of water containing 0.1 % formic acid, and mobile phase B was acetonitrile containing 0.1 % formic acid. The flow rate was set at 0.25 mL/min. Gradient elution was performed with initial conditions starting at 0 % B, held for 1.25 minutes, increasing to 10 % B at 6.00 minutes, 25 % B at 8.00 minutes and 75 % B at 10.50 minutes, followed by a wash step to 95 % B until 11.50 minutes. At 11.50 minutes, the flow was returned to initial conditions (0% B), allowing for 3.50 minutes of reequilibration time. A 10 µL injection volume was performed. Mass spectrometry was performed on a Bruker Impact II QTOF-MS (Bruker, Bremen, Germany) with electrospray ionisation operated in positive and negative mode. The capillary voltage was set at 4500V in positive ionisation mode and 3500 V in negative ionisation mode. Drying gas was set at 10 L/min, gas temperature was 220 ºC, nebuliser pressure was 2.2 bar and the end plate offset was 500 V. MS1 scan rate was set at 12 Hz. Auto MS/MS was enabled, with 4 precursors automatically selected and data collected at a scan rate of 25 Hz per scan cycle, resulting in a total scan cycle time (MS1 + auto MS/MS) of 0.2 seconds. An internal calibration was performed by injection of 5 mM sodium formate solution in water:isopropanol (50:50 v/v) at the beginning of every run. Mass spectrometric data were collected with Compass HyStar 5.1 and O-TOF Control version 5.2. Data were reviewed using Compass DataAnalysis 5.2 (Bruker Daltonics, Bremen, Germany) and pre-processed in Metaboscape 2022 B. Multivariate statistical analysis was performed using SIMCA® 17.0 (Sartorius AG, Göttingen, Germany).

NMR spectroscopic measurements of MH-Ox and MH-Bm media.
(a) 1D 1 H with solvent pre-saturation for the MH-Bm medium. The aromatic region (left) is scaled up by a factor of ten compared to the aliphatic region (right) of the spectrum. (b) 1D 1 H with solvent presaturation for the MH-Ox medium. The aromatic region (left) is scaled up by a factor of ten compared to the aliphatic region (right) of the spectrum. (c) Direct comparison of the two growth media MH-Bm (black) and MH-Ox (red) focussing on the proton NMR regions that showed major differences in peaks and/or peak intensities. Corresponding structures of the molecules or moieties are given next to the peak(s) in question and the proton which yields the peak is presented in bold. The structures (from left to right) are: Adenine, Guanine moiety containing compound (unknown), Uridine, Uracil, Glucose and TRIS.  Table 10). Reference Standard (10 µg/mL) Mean, SD and p values for data in Fig. 4A. Values are mean ± SD for three biological replicates determined with Etest strips on MHF-Bm agar. A two-tailed, unpaired Student's t-test was used for comparison of groups. SMX  TMP  SXT  SMX  TMP  SXT  SMX  TMP  SXT   NS5437 1.333 ± 0.289 0.250 ± 0.000 0.023 ± 0.000 1.667 ± 0.289 0.250 ± 0.000 0.023 ± 0.000 0.230 1.000 1.000 NS5437::thfT 3.667 ± 2.082 0.190 ± 0.000 0.023 ± 0.000 >1024 0.190 ± 0.000 0.105 ± 0.035 3.600x10 -9 1.000 1.408x10 -3

MIC (µg/ml) no THF MIC (µg/ml) with THF p value
Mean, SD and p values for data in Fig. 4C. Values are mean ± SD for three biological replicates determined with Etest strips on MHF-Bm agar. A two-tailed, one-sample t-test was used to determine differences from the maximum resolution of the SMX Etest assay (1024 µg/ml) for NS5437::thfT. A two-tailed, unpaired Student's t-test was used for comparison of NS5437 groups. THF and related compounds used for metabolic rescue of GAS strains in the presence of antibiotics.